We demonstrated the presence of physalaemin-like immunoreactivity in acid extracts of mammalian gastrointestinal tract and respiratory tract by use of an NH2-terminal specific antiserum to the amphibian peptide physalaemin. By radioimmunoassay the highest concentration occurred in rabbit stomach (ca. 100 ng/g dry weight) with levels of 1-15 ng/g in pulmonary tissue from numerous species. Immunohistochemical staining revealed specific immunopositive cells in Brunner's gland, in the duodenum and ileum of guinea pig, and in the colon of rat. Nerve fibers were also identified in the esophageal sphincter of rat. Gel filtration studies indicated that the immunoreactivity had a Mr =1700. This material was resistant to digestion by trypsin, alpha-chymotrypsin and CNBr. It eluted from alkylsilane columns in 35% methanol, whereas physalaemin appeared in 50% methanol. This material did not bind to Con-A. These data suggest that this immunoreactive material is a new neuropeptide.